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. 2015 Mar 30;43(8):4284–4295. doi: 10.1093/nar/gkv189

Figure 2.

Figure 2.

Activity and oligomers of Twinkle. (A) DNA unwinding activity of the three forms of Twinkle, expressed in SF9 insect cells (SF9, lanes 3 and 4), in E. coli (lanes 5 and 6) and in HEK mammalian cells (HEK, lanes 7 to 9, produced as previously reported) (25), was analyzed by monitoring the unwound ssDNA radiolabeled product of 60 bases from the M13(+) ssDNA plasmid. Lane 1 contains substrate; lane 2, boiled substrate; and the remaining lanes contain the indicated amounts of Twinkle (from 30ng to 100ng). Helicase activity of Twinkle was tested in the presence of UTP, except lane 8 for the HEK cells (lane 60ng -UTP). (B) Samples of Twinkle produced in insect cells purified by size exclusion chromatography were analyzed by negative staining EM. The two-dimensional class averages obtained by classification show the presence of hexamers, heptamers and open rings.